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Voltage-gated calcium channels swing open with passing action potentials, allowing calcium ions into the cell, and slide shut when the membrane depolarization has passed. Two papers this month suggest that these channels—particularly the long-activating L-type calcium channels (LTCCs)—are crucial to both axon regeneration in the peripheral nervous system and memory formation in the brain. In the online Current Biology, researchers from the Max Planck Institute of Neurobiology in Martinsried, Germany, report the solution to a long-standing mystery in neuroscience. They found that electrical signals, mediated by LTCCs, prevent axon outgrowth. And in the June 23 Journal of Neuroscience, scientists from the Technical University Munich, Germany, describe the role of a specific LTCC subtype, Cav1.2 channels, in fear-associated memory. Together the papers highlight novel roles and versatility of these membrane channels.

Close Channels for Axon Growth
The Martinsried researchers, led by senior author Frank Bradke, investigated the regeneration of axons in primary sensory neurons. These neurons, in the dorsal root ganglion, transmit sensations of body positioning (proprioception) and the feel of light touch to the brain. Instead of the usual axon-and-dendrites combo, they possess two long branches. One reaches out into the periphery, as far as the tips of the toes, to collect signals that travel up to the cell body. A second projection carries those signals from the cell body to the spinal cord, and up to the brain. This signal is unidirectional from the tips of the peripheral branches to the central nervous system.

The puzzle, which kept scientists wondering for decades, Bradke said, involves the phenomenon called conditioning. If the part of the nerve leading to the central nervous system is severed, it cannot regenerate. But if, prior to this central injury, the peripheral axon is crushed or severed, then the central branch does grow a bit. The peripheral branch injury somehow “conditions” the cell to regenerate after a central branch injury. “What the field is after, for quite some time, is to understand what is the signal,” Bradke said. “Could it be,” he wondered, “that it is simply electrical activity?”

First author Joanna Enes and colleagues first used cultured rat dorsal root ganglion neurons to test this idea. Normally, these cells will send out processes, or neurites, in culture. But the researchers soaked some of the cells in potassium chloride to mimic a constant state of action potential, causing membrane depolarization. These chronically activated cells sent out fewer neurites, and the ones they formed were shorter. And when the researchers used electricity to acutely stimulate neurons that were growing neurites, the growth halted upon stimulation. The authors concluded that membrane depolarization inhibits neurite outgrowth.

The authors reasoned that membrane depolarization triggers calcium influx, and there are several calcium channels that might be responsible for the effect. Using pharmacologic blockers to different channel types, they narrowed the field down to one responsible channel, the L-type Cav1.2 calcium channel. When they blocked LTCC activity, the growth inhibition from depolarization dropped by half.

The results explain some of the conditioning signal, but not all, said Jerry Silver of Case Western University in Cleveland, Ohio, who was not involved with the study. “It is only one part of the trigger,” he said. Bradke and his group are currently exploring possible effectors that could convert and transmit the electrical signal into something that would block outgrowth.

The results are surprising, Bradke noted, in that electrical activity is bad for regeneration. “It goes against a common dogma in the field,” Bradke said. “It may be good for many cells, but for our sensory cells, it may be that they use this electrical activity as a signal to know they are connected.”

Open Channels for Memory
The regeneration pathway is just one area where LTCCs are crucial. They are also needed for synaptic plasticity and memory formation, but researchers have not been certain which LTCC subtype is responsible for this job. Some studies have suggested the Cav1.2 subtype is the key to memory (Busquet et al., 2008), and the Munich researchers explored this possibility in the lateral amygdala, where fear-associated memories form. Leading the study were joint first authors Nicole Langwieser and Carl Christel and senior author Sven Moosmang.

First, the researchers treated brain slices with isradipine, which blocks LTCCs subtype Cav1.2, and found it prevented long-term potentiation. So they moved into animal studies, using a tone followed by mild foot shock in an associated fear paradigm. The animals freeze upon hearing the tone—if they remember the pain it heralds. Treating animals with isradipine before the tone-shock training blocked this memory formation, and the mice showed less freezing behavior 24 hours later than control animals that only received the drug vehicle.

The isradipine experiments indicated that CaV1.2 is involved in laying down memories. The researchers tried to confirm this using CaV1.2 conditional knockouts (cKOs). In fact, the cKO mice froze upon hearing the tone as often as control animals, leaving the researchers to seek alternate explanations for memory formation in the mutant mice. They considered that calcium-permeable AMPA receptors might fill the gap. To test this hypothesis, they treated brain slices from the CaV1.2 cKO animals with the AMPAR blocker PhTX. Sure enough, when the inhibitor was present, synaptic remodeling stopped.

“Calcium influx through Cav1.2 L-type calcium channels is critical for the formation of emotional memories,” Moosmang concluded in an e-mail to ARF. “However, if you lose the activity of these channels over a long time, major homeostatic changes are induced in the brain regions responsible for fear memory.” The researchers are working to understand the mechanism that allows AMPAR to stand in for the LTCC, and Moosmang speculated that the gene Art, known to alter AMPAR trafficking, might be involved in the switch.

These two studies suggest that LTCC modulators might someday be components in cognition enhancers as well as axon-regenerating cocktails. LTCC blockers are already available to treat heart problems, Silver noted. However, Moosmang wrote, “Much work has to be done to really translate this idea into the clinic.”—Amber Dance